Morphological and Molecular Phylogenetic Data Reveal a New Species of Primula (Primulaceae) from Hunan, China

A new species of Primulaceae, Primula undulifolia, is described from the hilly area of Hunan province in south-central China. Its morphology and distributional range suggest that it is allied to P. kwangtungensis, both adapted to subtropical climate, having contiguous distribution and similar habitat, growing on shady and moist cliffs. Petioles, scapes and pedicels of them are densely covered with rusty multicellular hairs, but the new species can be easily distinguished by its smaller flowers and narrowly oblong leaves with undulate margins. Molecular phylogenetic analysis based on four DNA markers (ITS, matK, trnL-F and rps16) confirmed the new species as an independent lineage and constitutes a main clade together with P. kwangtungensis, P. kweichouensis, P. wangii and P. hunanensis of Primula sect. Carolinella.


Introduction
The genus Primula L. can be explicitly recognized by its perennial habitat, basal rosette of leaves, heterostylous flowers with obvious tube and capsular fruits, with rare exemptions (with homostylous flowers, which have only one flower type, with anthers and stigma at the same level) [1,2]. The heterostyly is a distinctive feature, with species possessing short-styled thrumtype flowers with the style included in the corolla tube, and the long-styled pin-type flowers with long style and the stigma presented at the throat of the tube. Recent molecular phylogenetic studies indicated that Primula (with Dionysia Fenzl, Sredinskya (Stein) Fed., Dodecatheon L., and Cortusa L. nested) is a major monophyletic clade of Primulaceae, while several genera traditionally included within the Primulaceae (e.g. Samolus L. and Lysimachia L.) have been transferred to the Theophrastaceae or to the Myrsinaceae [3][4][5][6][7]. This genus consists of more than 500 species, mainly distributed in the temperate zone of the Northern Hemisphere [2,8,9]. The modern center of diversity of Primula is in southwestern China (particularly in western Sichuan, eastern Xizang, and northwestern Yunnan), including approximately 300 species [1,8].
Recently, some molecular phylogenetic analyses focusing on Primula have found that most of the subgenera and sections in the traditional subgeneric classification of Primula were nonmonophyletic [5,7,13,14]. Some of the diagnostic characters, such as the calyptrate capsule, might have evolved more than once in Primula, even in Primulaceae [14,15]. The subgen. Carolinella was nested within subgen. Auganthus and differentiates into four separate lineages [14,15]. However, a formal taxonomic treatment of the genus Primula based on the latest molecular phylogenetic studies has not yet been proposed [13]. The traditional morphological characters (including the calyptrate capsule) are still the main evidence to establish subgroups of Primula in floras and taxonomical studies [16].
In the early 2014, during field expedition in southwest Hunan, China, one of the authors fund a distinct Primula species with undulant margin of leaves. At first sight, this species is similar to P. kwangtungensis, and is also easily confused with some variants of P. obconica Hance (belongs to sect. Obconicolisteri). Further morphologic investigation of both flowering and fruiting individuals turned out that it represents an undescribed taxon. Evidenced by the unique calyptrate capsule among sections in Primula, it is supposedly affiliated to the sect. Carolinella. Thus, here we not only make a morphological comparison with related species but also conduct a phylogenetic analysis to determine the systematic position of the new species.

Ethics statement
All samples employed in this study are not endangered nor protected in the sampled area, and none of the sampled locations are privately owned or protected by any law. No specific permits are required for the present study.

Nomenclature
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Taxon sampling
The present phylogenetic analysis was based on the framework of Primula, with subgen. Auganthus as focal group, with reference to recent molecular phylogenetic studies [4][5][6][13][14][15]. We downloaded 311 sequences for 66 species of Primula from GenBank. In addition, we sequenced five species including the new species and the recently described P. hunanensis. Totally, the ingroup included 342 sequences (86 accessions) of 68 species, representing all main sections of the genus Primula and all nine sections of subgenus Auganthus established by Richards [9]. Two species of the related genera to Primula, i.e. Androsace sublanata Hand.-Mazz. and Soldanella alpina L., were selected as outgroups following previous phylogenetic studies [5,6,13,23]. Voucher information and GenBank accession numbers are listed in S1 Table. DNA extraction and sequencing Total genomic DNA was extracted from silica gel-dried plant leaves following a modified CTAB protocol of Doyle & Doyle [24]. The nucleotide sequences of the nuclear ribosomal internal transcribed spacers (ITS, including ITS1-5.8S-ITS2) and three chloroplast regions (matK, rps16 and trnL-F), which were employed to infer phylogenetic relationships of Primula in previous studies, were analyzed in this study. For ITS, the primers, PCR amplification and sequencing conditions followed Liu et al. [14]. For chloroplast regions, the primers, PCR amplification and sequencing conditions followed Yan et al. [15], except the matK region of two samples (see S1 Table for details) that followed Yan et al. [25].

Sequence alignment and phylogenetic analysis
SeqMan (DNASTAR Inc., Madison, WI, USA) was used to assemble sequences. Multiplesequence alignment for each marker was made using Clustal X 1.83 [26], and then minor manually adjusted using Se-Al version 2.0a11 [27]. Following the treatment of Liu et al. [14], combined phylogenetic analysis (including all the four datasets) was carried out using maximum parsimony (MP) and Bayesian inference (BI).
MP analysis was conducted using heuristic searches of random taxon addition for 100 replicates through PAUP Ã version 4.0b10 [28], and tree bisection-reconnection (TBR) branch swapping with no limit to the number of trees saved. All characters were weighted equally and gaps were considered as missing data. Relative support for individual clades was evaluated by Bootstrap analyses, which was conducted from 1000 replicates of heuristic search with TBR branch-swapping and saving multiple trees [29].
Bayesian analysis was carried out using MrBayes version 3.2.1 [30]. Best-fitting nucleotide substitution model for the combined data matrix was selected by ModelTest version 3.7 [31]. The Bayesian Markov Chain Monte Carlo (MCMC) algorithm was run for 2000000 generations under a GTR + I + G substitution model, with sampling of trees every 200 th generation. The initial 25% of sampled trees were discarded as burn-in after checking for stationarity and convergence of the chains with TRACER v1.6 [32]. The 50% majority-rule consensus tree was calculated from remaining trees with nodal support summarized as posterior probabilities (PP).
Bootstrap values ! 80 and Bayesian posterior probabilities ! 0.95 were considered as robust support.

Morphological observations
We observed and measured both fresh and pressed specimens of the new species to obtain the accurate description. All the quantitative characters, like the diameter of corolla, which were easily changed from fresh to dry, were measured from forty fresh individuals (with half thrum flowers and half pin flowers) in the field, randomly. Other qualitative characteristics, like the texture of leaf, which were stable, were observed from ten pressed specimens. Specimens of the related species, i.e. P. henryi, P. chapaensis, P. partschiana, P. rugosa, P. kweichouensis, P. kwangtungensis, P. wangii, P. hunanensis, P. levicalyx and P. calyptrate, were checked and a morphological comparison was made with the new species. The occurrences of the main diagnostic characters (inflorescence types and calyx shape) of the members of P. sect. Carolinella were displayed on the resulting phylogenetic tree. The information of the examined specimens can be found in the S1 Text.

Phylogenetic analysis
Aligned length of the combined dataset (ITS + matK + trnL-F + rps16) was 4655 bp, of which 1901 sites were variable and 1253 were maximum parsimony informative. The majority consensus tree of the Bayesian analysis of the combined dataset is shown in Fig 1, and the topology was congruent with that of MP analysis. The combined matrix and the majority consensus tree were deposited in TreeBASE; study number S19166 (http://purl.org/phylo/treebase/phylows/ study/TB2:S19166).
Species of sect. Carolinella were divided into four separate clades (Fig 1; clades A, B, C, D). Two samples of P. chapaensis deviated and were deeply nested within subgen. Auganthus (Fig  1; clade A). Other examined species of sect. Carolinella fell into three phylogenetically independent lineages (Fig 1; clades B, C, D). The clade including all the samples of P. partischiana and P. rugosa was first diverged from the remaining group of subgen. Carolinella (Fig 1; clade D). Then the clade of P. calyptrata was differentiated as an independent lineage (Fig 1; clade C). The new species together with P. hunanensis, P. wangii, P. kweichouensis and P. kwangtungensis, formed a clade and became the sister to the majority members of subgen. Auganthus (Fig 1; clade B). All the three samples of the new species formed a robust monophyletic group (Fig 1; clade B).

Diagnosis
At first sight the new species seems to be some variants of P. obconica Hance, but the calyptrate capsule clearly shows that its true affinity is with the species of section Carolinella, and the most closely related species is P. kwangtungensis, but the new species can be easily distinguished from P. kwangtungensis by its smaller flowers and narrowly oblong leaves with undulate margins.

Description
Herbs perennial, efarinose; rhizome very short, with numerous fibrous roots. Leaves in a rosette; petiole slender, 2.5-5.5 cm long, half the length of the blade to almost as long as it, densely covered with rusty curled multicellular hairs; leaf blade oblong to oblong-elliptic, 2.5-6.5 × 1.2-4 cm, papery when dry, abaxially sparsely pilose along main veins and veinlets, adaxially sparsely pilose along midvein, otherwise glabrous, apex rounded, base subrounded, sometimes slightly cordate and asymmetrical, margin undulate, with irregular wavy undulations, ciliate; lateral veins 5-6 on each side of midvein, c. 45°angled to midvein. Scapes 2 to many per plant, 2.5-8.5 cm long, together with pedicels densely covered with rusty multicellular hairs; umbel 3-5-flowered, solitary or with a second whorl of flower; bracts linear, 3-5 mm long, less than 1 mm broad, apex acute, more or less pilose at base; pedicels slender, 1.5-2 cm long, lengthening to 2.5 cm in fruit. Flowers distylous. Calyx campanulate to tubular-campanulate, 5-5.5 mm, split to a third of its length; tube pilose outside; lobes ovate, c. 1.5 mm wide, apex acute, glabrous. Corolla pale violet-purple, annulate, limb c. 1.5 cm wide, lobes obovate, c. 7 × 4 mm, bilobed, lobules entire; corolla tubes c. 9 mm long, thrum flowers with stamens inserted towards apex, style slightly exceeding the calyx; pin flower with stamens inserted on the middle of the corolla tube, slightly exceeding the calyx, style equaling the corolla tube. Capsule c. as long as the persistent calyx, with a fragile wall, at first opening by a thicker lid.

Distribution, habitat and phenology
The new species is presently known only from the type locality in Dong'an Xian of Hunan Province (Fig 4). It grows on shady and moist cliffs of karst landform, alt. 348 m. Flowering in March to April.

Etymology
The species epithet "undulifolia" refers to the leaf margin of the new species with wavy and shallow undulations.

Conservation status
The new species is presently known only from the type collection with two small populations. The habitat adjoins to the farmland, and become fragile due to deforestations and farming activities. Based on current information and according to IUCN red list criteria [33], its conservation status is evaluated as 'Critically Endangered' (CR) (B1abiii).

Taxonomy and phylogenetic relationships
Based on the similar habit and vegetative characters, sect. Carolinella was considered having affinity with sect. Obconicolisteri, but can be distinguished by its calyptrate capsule [19]. The new species also seems as a variant of P. obconica by the leaf morphology at first sight. In this study, traits of calyptrate capsule, tubular-campanulate calyx and rusty indumentum, as well as the molecule phylogenetic result all reveal that P. undulifolia should be affiliated to sect. Carolinella defined by Hu [19], and are closely related to P. hunanensis and P. kwangtungensis. While species from sect. Obconicolisteri generally have a broadly campanulate calyx.
The non-monophyly of sect. Carolinella shown in this study has been verified by the recent phylogenetic analyses [14,15]. All the examined species of sect. Carolinella are deeply nested within subgen. Auganthus and divided into four clades (Fig 1; clades A, B, C, D). We confirmed the systematic position of the new species P. undulifolia, which clusters with P. hunanensis, P. wangii, P. kweichouensis and P. kwangtungensis (Fig 1; clade B). Morphologically, this clade can be distinguished from other clades of sect. Carolinella (Fig 1; clades A, C and D) by the umbel with heterostylous flowers (Fig 1B'). In contrast, species of clade A and D have abbreviated raceme (Fig 1A' and 1D') and clade C has superimposed umbel with homostylous flowers (Fig 1C'). Additionally, calyx lobes of clade B are ovate-triangular (Fig 1B"), while that of clade A are ovate and veined (Fig 1A"), clade C are ovate (Fig 1C"), clade D are triangular and apex acuminate to caudate (Fig 1D").
Although, the phylogenetic relationship of clade B (Fig 1) is unresolved, all the three accessions of the new species P. undulifolia constitute a robust monophyletic group. Morphologically, P kwangtungensis is closely related to P. undulifolia with the same traits of indumentum and calyx, and it is sister to all the other species (including P hunanensis, P. wangii, P kweichouensis and P. undulifolia) of clade B (Fig 1). Meanwhile, except for P kwangtungensis, the remaining species of clade B (Fig 1) are distinct for each other: pedicel and calyx of P hunanensis are glabrous, leaf blade of P wangii is more or less cordate at base, calyx of P. kweichouensis split to 2/3 of its length and leaf margin of P. undulifolia is undulate. Otherwise, the geographical distributions of all five species, included in clad B (Fig 1), are non-overlapping (Fig 4).

Diversification of Primula in subtropical mountains
Previous studies [2,8,16,25] indicated that the geographical distribution of the genus Primula is an outstanding example of high diversity and endemism. As a whole the distribution of the genus is worldwide, but with most of the species concentrated in a narrow area, stretching from East Himalaya to the high mountains of western provinces Yunnan and Sichuan of China. Of the 300 species in China, more than 80% occur in this area.
Meanwhile the recognized clades comprising the new species (Fig 1; clades A-D) in the present study represents a few outliers that are found in the subtropical mountains, with a distributional range from SE Yunnan and W Guangxi along Nanling mountain extending to N Guangdong, in a narrow (c. 250 km wide) strip of area, between latitude 23°N to 26°N, longitude 103°E to 113°E (Fig 4). This region harbors almost all the species of sect. Caronlinella (except P. intanoensis distributed in Thailand and P. cardioeides distributed in Vietnam) and a few members of sect. Monocarpisae (i.e. P. malacoides Franch. and P. cavaleriei Petitm.) and sect. Obconicolisteri (i.e. P. barbicalyx Wright, P. obconica Hance and P. apicicallosa D. Fang). Compared with the alpine species, the subtropical species of Primula have wide blade and conspicuous petiole. These species also demonstrated highly endemism and abundant diversity in the special ecological niche of Danxia landform and karst landform of this stripe. The morphological and distributional range of the new species suggest that it is closely related to P. hunanensis and P. kwangtungensis, all adapted to subtropical climate, having adjacent ranges and similar habitat, growing on acidic soil of Danxia landform or karst landform.
Supporting Information S1 Table. Species, voucher information and GenBank accession numbers. (XLSX) S1 Text. The examined specimens of species related to the new taxon. (DOCX)